Of the 180,000 new cases of breast cancer that are diagnosed annually in the United States, approximately 140,000 will have no clinical evidence of metastatic disease. Almost all of these patients are candidates for adjuvant chemotherapy, hormonal therapy, or both. Yet, 70,000-75,000 of the 100,000 patients without axillary node involvement and 12,000-15,000 of the 40,000 patients with axillary node involvement, would remain free of disease for the remainder of their lives even without adjuvant treatment. If the 90,000 patients who are actually cured with local therapy could be identified with confidence, they could be spared unnecessary treatment, and the health care system could be spared an unnecessary expenditure that can be estimated to exceed 500 million dollars annually (assumed treatment cost per patient in the range of $5,000-$10,000).
Although many statistically significant prognostic factors have been identified in breast cancer, no single factor has been found to date that can separate patients who are likely to relapse from those who are likely to remain disease-free cleanly enough for making clinical therapeutic decisions. For example, the presence of tumor cells in the bone marrow has recently been found to correlate very well (P<0.001) with subsequent recurrences that involve bone, but not with loco-regional or purely visceral recurrences (1). One might wish to combine prognostic factors empirically to achieve a better separation between patients who are likely to develop tumor recurrences from those who are not; indeed, the combination of axillary nodal status and the presence or absence of tumor cells in the marrow appears promising (1). However, the empirical approach is by its very nature haphazard and inefficient, and seems unlikely to produce results that would be universally accepted as definitive.
The premises underlying the invention herein are that progress in the application of prognostic factors in breast cancer will ultimately depend on the intelligent use of such factors in combination, that the most robust combinations of prognostic factors are likely to be those that are based on biological relationships at the molecular level.
This approach has been 1) to determine critical sequences of genetic evolutionary changes in breast cancer that are responsible for increasing tumor aggressiveness, in order to establish how far a given tumor has progressed in its genetic evolutionary sequence, 2) to determine if a given tumor has undergone the critical steps in the sequence that are necessary for cellular acquisition of the capacity to metastasize, and 3) to apply this information clinically for purposes of prognosis and adjuvant treatment planning.
The underlying principle that guided the work described herein was that the increase in tumor aggressiveness that accompanied tumor progression was the result of an accumulation of genetic abnormalities within individual cells. This was confirmed by studies in which multiple correlated measurements on each cell in each tumor sample were performed by means of multiparameter flow cytometry (FCM), by multiparameter fluorescence in situ hybridization (FISH) studies, and, more recently, by laser scanning cytometry (LSC). Patients have been followed prospectively to assess the biological consequences and clinical outcomes of the patterns of intracellular geno-phenotypic abnormalities that were found in the cells of their primary tumors at the time of surgery. Early studies showed that the accumulation of aneuploidy, Her-2/neu overexpression and ras overexpression in the same cells (triple positive cells) was of prognostic significance in breast cancer (2). These ongoing studies have recently been updated, and the prognostic information conveyed by the intracellular accumulation of these three abnormalities has been found to be of even greater statistical significance after up to 10 years of follow-up.
It has been found that the commonly observed pattern of aneuploidy, Her-2/neu overexpression and ras overexpression was characteristic of approximately two thirds of p53-dysfunctional, non-lobular breast cancers (chiefly infiltrating ductal tumors), whereas the characteristics of lobular tumors (representing ˜10 percent of the total), and those of the remaining one third of the p53-dysfunctional, diploid non-lobular breast cancers were quite different (3-5). Studies have also indicated that within individual tumors the development of p53 dysfunction generally occurs before the development of Her-2/neu overexpression, and that ras overexpression is a late event (4). Based on these findings, it was concluded that the majority of non-lobular breast cancers follow an evolutionary pathway in which wild type p53 function must be abrogated before sustained receptor tyrosine kinase-induced, ras-mediated mitogenic signaling can proceed unimpeded. The fact that p53 abnormalities, aneuploidy, Her-2/neu amplification/overexpression, and ras overexpression can all be found in ductal carcinomas in situ (DCIS), a preinvasive and pre-metastatic stage of breast cancer, suggests that these changes herald subsequent evolutionary pathway-specific abnormalities that are more directly responsible for the acquisition of metastatic potential.
A major factor that constrained the clinical application of these findings in the past was the presence of false negative tumors (tumors that did not contain detectable proportions of triple positive cells, but recurred nevertheless). The false negative rate, which was in the range of 10 percent, had to be reduced substantially before this approach could be used to identify specific patients who are at such low risk for recurrence that adjuvant therapy can safely be withheld. In studies, the false negative tumors could be assigned to one of four groups, based on the patterns of intracellular abnormalities that they contained. An orderly strategy has been adapted for identifying combinations of molecular abnormalities within each of these groups that would distinguish patients at high risk for recurrence from those who are at low risk. This strategy is at the heart of the technique herein, since it assures that one can reduce the prognostic false negative rate progressively, until a level is reached that is acceptable for clinical application, no matter what that level might be. Then, aggressive therapy can be applied to patients at high risk for the presence of occult disease or micromatastesis. While much of the work to date has been carried out in breast cancer and lung cancer, the principles underlying this approach are applicable to all types of human solid tumors.